Beckwith-Wiedemann Syndrome (BWS) is the paradigm of cancer-predisposing disorders caused by an epigenetic mechanism, and it serves as a model for a general understanding of cancer epigenetics. Patients with BWS have an 800-fold increased risk of Wilms tumor (WT) and other embryonal cancers of childhood, as well as birth defects including increased size, organ overgrowth, and midline closure defects. We earlier mapped BWS to a region of 11 p15 containing an unidentified WT gene (WT2) lost in WT and common adult cancers. In the first 31/2 years of this MERIT award, we have performed the first epigenotype-phenotype study of any disorder, showing that BWS is a contiguous gene syndrome with the genes H19 and IGF2 causing cancer, and LIT1 and p57^p2 causing birth defects. We made the first link between any disorder and assisted reproductive technology (ART), showing an association with epigenetic changes in LIT1 in BWS. We also identified the first chromosomal microdeletion in BWS, identifying an imprinting element controlling T^pz and other genes, and a novel chromatin insulator in LIT1. Finally, we identified several potentially critical novel genes, including a silence information regulator (SIRT3) localized to mitochondria, and a candidate WT2gene. This previously unappreciated imprinted gene encodes a protein localized to the nucleolus, its expression is lost in WT with loss of heterozygosity or loss of imprinting, and it suppresses the growth of WT and other tumors. In the MERIT extension, we will identify additional microdeletions in familial BWS and the regulatory elements and genes within them. We will investigate the mechanism of ART-induced epigenetic defects in BWS through in vitro culture and fertilization of mouse gametes, and the effect of epigenetic defects on cancer incidence in the offspring. We will investigate the molecular basis of the 30% of BWS patients without methylation changes, as well as idiopathic hemihypertrophy patients, using new chromatin immunoprecipita- tion approaches developed in the current grant period. We will investigate the relationship between genotype and epigenotype in families. We will continue to identify new genes and regulatory elements in BWS, using epigenomic and functional approaches, including a putative enhancer for p57KIP2 and a gene for hypoglyce- mia. We will perform genetic and functional analyses of the candidate WT2 gene and another gene, identi- fied in the current grant period, that may mediate LIT1 function. These studies will include in vivo mouse models, biochemical studies of nucleolar protein binding, and identification of binding partners, including a novel insulator protein that binds to LIT1. This work should continue to provide pioneering insights into the genetics and epigenetics of cancer, as well as the role of environmental and developmental miscues in epigenetic disruption, with broad implications for understanding the mechanism of human cancer.